Report of Dr. Avex with Drs. Stillman, Goebel, Dubp_s, _- ____-_-_cI-- ------------ Francis Rellx --,,,I,-- A-aa!Ers LGood"er and Allowax I----I----4 1. I. The decomposition of the Capsular Polysaccharide of Type III Pneumococcus by a Bacterial Enzyme. 1. Methods for obtaining potent, non-toxin preparations of the enzyme. 2. The protective action of the specific eneyme against Type III Pneumococcus infection in mice. II. Isolation of other mlcroorganlsme decomposing the capsu- lar polysaccharides of different types of Pneumococcus. III. Chemo-immunological studies on Carbohydrates. 1. The determination of the molecular size of the cap- sular polyeaccharide of Type III Pneumococcus. 2. The specific carbohydrate of two strains of Pfeiffer's bacillus. 3. The somatic Carbohydrate of Pneumococcua. 4. Studlee on synthetic carbohydrate derivatives, a) Syntheele of , lated as glucose, -oh hydrolysis.`. At `th/j kuggestion of `Dr. " Franoie; I, _ a phosphorous an'alyeia wae"mad& on this' substance" $t Dr. Heidelberger. at. the,`Preabyte+l&i Hospital. -The car- bohydrate contains organically bound phosphorous to the ex- tent of approximately 3.3 per cent. Re have also found this material to contain about 5 per cent of nitrogen. The car- bohydrate is hydrolyzed by nitrous acid in the cold, with an accompanying loss in specificity. We have attempted to split off the organic phosphorous of this carbohydrate by means of a powerful phosphatase, but the enzyme causes no liberation of inorganic phosphorous. At present sufficient quantities of this material are being prepared for Dr. Francis in order that he may carry out certain clinical etudies. 4. Chemo-lmmunolo&cal studies on synthetic carbo- -- -_c-------- ----------- Qdrate derivatives. e-e- In previoue reports we have described the method for the preparation of synthetic carbohydrate (hexoee)-protein antigens, ae well ae the preparation of a polysaccharlde-protein antigen which elicit specif lc immune responses when inJected into animale. From these studies we have learned f lrst, that carbohydrates, both eimple and com- plex, can be rendered antigenic when they aLpe coupled to a protein, and second, that the specif lc immune response (in the case of heXos88) is determined by the configuration of the heroee molecule. ,Tn the case of large molecules, such as the Bpecif ically reaotlng baaterial polyaaccharldee, the ultimate immune responee is such that the induced antibody will not only agglutinate microorganisms from which the car- bohydrate was derived, but will actually confer passive pro- tection on mice infected with an homologous type of organism. We are inclined to believe that in order to secure immunity against a disease in which the chief Immune response is de- pendent upon the elaboration of anticarbohydrate antibodies, it Is necessary only to have a8 the antigenic agent, a com- bined carbohydrate, of an exact and correct chemical confi- H 106 guration, capable of stimulating the formation of these an- tibodies. In a molecule as complex as that of Type III Pneu- mococcus polysaccharide there are naturally many factors which enter into the orientation of its ultimate specificity4 We know, for example, that the molecule is studded with highly polar carboxyl groups. We know that there are three free hydroxyl groups per unit of hgxose, and we know that the molecule is a polymer of an aldobionic acid (glucurono- glucose). We do not know, however, the mode of linkage of one aldobionic acid unit to the next; we do not know whether the glucurono-glucose is an j: or $ glucuronoside, nor do we even know If an A, sugar can give rise to a different antibody than its !:I homologoue. However, these fundamental problems in the under- standing of the spec$ficity of fmmtinological reactions in the case of carbohydrates are not entirely unapproachable, for we have at our dieposal methods based on synthetic chem- ietry, whereby we can analyze the influence, not only of in- termolecular stesechemical relationships, but the influence of polar groups In the carbohydrate molecule as well. ,a) The synthesis of 4 Jara-amino phenol gluco- I_ -------c- --------- ------ ---- side ---z We have demonstrated in previous work that the alter- ation of tho stereo-chemical configuration of one carbon atom in different hexoses, suffices to elicit totally differ- ent and specific antibody response. Our next problem is to __--__ --- :31-t H 107 ascertain whether the 4 and @ glucosides of the same hex- ose would give rise to separate and distinct antibodies, or merely to antibodies showing but slight differences. ye have already synthesized the v-& o-nitro phenol glucoside of glucose. The homologous (I( glucoside has been synthesized in the following manner. C HOH c H0130CH3 H-,C-Cl /' / I 1: , /: C EOH / ,/' I C HOCOOH3 I' b HOCOC3 0, 6 HO2 (CH,CO),O $ i /I I ----- "' 1: HOB -3 `\ C HOCOCH3 4 PC$ Q( \ 1 IHOCOX 1, j \F HOCOC~?; ----+ . I \F HoCoCH3 + 7" t H '4 H C II203 ; H OCOCH3 b :i20COCH3 Glucose B-pentacetyl glucose 2 trichloracetyl 3,4,6 triacetyl $-glycosyl chloride ,?oC13 H- F - Cl ,I" 3 HCl I C HOE "\ NH3, / / I \ 3 PC13 HOCOCH3 + Ago `;302 CH3COCl CH30H \ F '7 HOCOCH3' -4 b (Walden inversion) 'II I . CH2OCOCH3 3,4,S trlicetyl-' ' ' ' " "3;4,6'triacetyl ':? n,~l~,~.nr?vl chloride p-nitro phenol o * glucos5dL; ;I \ J-~".." ,JU - ----- - - NO2 `z;; - 0 - ;? - H /, / CHOH bHOCOCH3 NH3 0,. ',. I I' `\ \ CHOCOCH3 CH2OCOCH 3 NON cZj - 0 -,,v - H ./ /! ' CHOH I 0:' &OH \ `\ i p-nitro \ 'l CHOH phenol" b glucoside . H I CH20H The nitro phenol glucosido has not as yet been reduced to the amino homologus. The 4 nitro phenol glucoaide analyses correctly for carbon and hydrogen. It melts at 220o and has an optical rotation of +233,9O whereas its isomer, the (3 - glucoside, melts at 164O and rotates the plane of polarized light - 79.6". We intend to reduce the L% compound catlyt- ically, to couple it to protein and to immunize animals and finally to compare the antibody thus produced with those ob-. tained by immunization with the (4 -glucoside-protein complex. b) Studies onglucuronic acid. --- We have spoken above of the occurrence of gluct%%`onic acid in the Type III Pneumococcus specific polysaccharide, and we have indicated that the carboxyl groups remain free in the intact molecule. Not only have we found glucuronic acid in this specific car- bohydrate, but we have found it in every bacterial polysac- charide investigated in our laboratory. Furthermdre; we have found it in' gum acacia, and we have shown that gum aca- cia will react with antipneumoco'cbuh Types IT `and III aera in dilutions dd'high'ae 1:6,000,000 `of the carbohydrate. It seems, therefore, that this Important constituent o'f so many spocif ic polysaccharides must play an important immunologi- Cal role. It is now our belief that the highly polar car- boxy1 group of giucuronic acid, and its relationship to other polar groups in the carbohydrate molecule, exert a strong influence in the immune response. in order to test this conception, we have decided to determine whether there are 6". tGl3 H 109 differences in the antibodies produced by imclunization with protein complexes of e-p-amino phenol glucoside of glucose and those of I?-p-amino phenol glucoside of glucuronic acid. ___ . NH2 ._' - 0 -,d? - ii < 2 NH2 (--: - 0 - - H / ,' /# H9 OH , H + OH // .z I HO F H 0" HO i: H \ "\,H 0 OH -1 `-.... H ; OH . I `\C H ! C H20H y.. `W 0 OOH (3 -para amino phenol glucoside P -para amino phenol glucoside of glucuronic acid From these' two formulae one may see that the ster- eochemical relationship of these two molecules are exactly al Ike. They differ only in that the symmetrical sixth car- bon atom of the glucose glucoside, a primary alcohol group, is In the form of a polar carboxyl group in the glucurono- side. Since the other atomic groupings of these two com- _,II : pounds are '&actly.aiike;' atiqe -iti this one respect; It is- possible `to determine directly the influence on the immune 9 . ..(.* ; response of the eubatftution of an acid grouping in a hexose ,p "L r molecule. > ts The'problem of syntheeizing the para-amino phenol glucoside of glucuronic acid is by no means simple. It I.0 difficult to secure large quantities of the sugar acid, and the chemistry of the acetyl and halogen acetyl derivatives from which these glucosides can-be prepared, la unknown. The method of procedure is as follows:- Glucuron- ic acid was prepared by feeding 5 grams of borne01 daily to each of 6 dogs. The animal organism conjugates the bornsol and excretes it in the form of a glucuronic acid glucoside which can be quantitatively recovered from the urine as a crystalline zinc salt. Between 20 and 30 grams of zinc borne01 glucuronic acid can be recovered daily from 6 dogs. The zinc borne01 glucoside is treated with sulfuric acid to split off the zinc, yidlding the borne01 glucoside of glucuronic acid. The latter is then hydrolyzed with 0.2 normal sulfuric acid, and the insoluble borne01 is separ- ated from the solution of glucuronic acid. After removal of the sulfuric acid by BaC03 the glucuronic acid can be crystallized as a snow white product. The chemical manipu- lation can be easily carried out, and the ultimate yield of this rare sugar repreeents nearly 90 per cent of the source material, The sugar \can be converted to its lactone by crya-, tallization from acetic acid. ,;C HOH GHOH / /' 1 C HOH / / \ OHOH o/' H '.\ "l\ 8 -HOH glucuronic acid glucuron :3 5 4 H 111 This derivative has been acetylated and J( and (3- triacetyl glucuron have been separated by fractional crystal- lization from alcohol. Neither the o( nor the (3 -triacetyl glucuron will yield a stable bromo derivative, but we have secured from both derivatives a stable diacetyl chloroglucuron. : 7 / HOH gl i: HOH / .I' I I (CH3CO)$ H - C\ --- \ "\ `,B 8. C HO x `\ `\., AH `/o / C." / j /`H 7 / OCOCH3 glucuron t riacetyl glucuron 1-chlor diacetyl glucuron ,.@iOCOCH3 `ri -,Q - Cl Though yet obtained in a crystalline state, these halogen acetyl derivatives give correct analyses. The syn- thesis has not as yet progressed beyond this point; but the , preparation of these two chloro diacetyl glucuron deriva- tives now make possible the completion of the synthesis of the para,amino phenol glucoside of glucuronic acid,!whlch we hope to accomplish in the near future, o : ,, ,,,:: ~ . IV. The Chemical Nature of the Tyne-specific ,. -: I. Capsular Antigen of Pneumococcus. (Drs. Avery ahd Goebel). The importance of determining the ch'emlcal nature of the type-specific antigen of Pneumococcus becomAs evident when one considers that the protective `substances in anti- pneumococcus serum are precisely those antibodies which are produced in response to this particular antigen. By speci- ----- 3,-l. 6 B 112 fit union with these antibodies, the capsular substance of: the virulent Pneumococcus bocomee so alterod that the bac- tcrial cell as a nhole is rendargd susceptible to phagocy- tosie. And, so far as our knowledge at present goes, the therapeutic effect of antipneumococcus serum is largely, if not exclusively, dependent upon the presence of these type- specific antibodies. Indeed, the application of serum thera- py in Pneumococcue today rests upon our knowledge of these type-specific relationships. It would appear, therefore, that an understanding of the chemical nature of this impor- tant antigenic constituent of the pneumococcus might contri- butemuch to the improvement and perfecting of methods useful in the production of potent antisera. Interestingly enough, al. though the exact nature of this antigen itself eludes us, we have considernblo knowledge of one of its component parts, namely, the typo-specific CEQ sular polysacchartJde+ For instance, we `knon that thie specific carbohydrate is given off from the bacteria growing . in the iocue of disease, gnd cir.culatee in soluble form in the .body fluids;. that, in thie form,it still retsins its capa- * * city to unite with and thus, to neutralize the immune substan- , ces of the serum; that even in minute amounts it inhibits phagocytosis, and that because of these reactions its pre- sence tends to interfere with the normal processes of recov- ery from the disease. We know also something of the oheml- stry and immunological specificity of this capsular substance. We have succeeded in chemically identifying a polysaccharide in the capsular substance of each of the different types of Pneumococcus and have found in each instance that the carbo- hydrate is as chemically distinct as it is serologically specific for each type. We have demonstrated the remark- able specificity of these carbohydrates in the phenomenon of anaphylaxis and in the reactions they incite in the skin of convalescents from pneumonia, we have evidence that these specific carbohydrates, when separated from the bacteria, still retain unimpaired the property of combining with anti- bodies, but lose more or less completely their power to in- cite the formation of these same antibodies when injected alone into the animal body. This very fact indicates that in their native etate, these specific polysaccharidcs exist in the cell not as hgtens but as part of a more complex antigen from which they may be more or less readily disso- ciated either bforo or after the Injection of the whole antigen into the animal .body. The rate and extent to which this dissoc%&tion ocour~ apparbntly varies with the dlffer- ent types. The theory of antigenic dissociation and its re-' latlon to the production of specific sera have been discuas- ed in a preceding rbport. It is referred to here merely to emphasize the importance of knowing the &emical n8tUr8 of the typ843p8CifiC antigen, and to point out again the prac- tical bearing which such knowledge might have upon the prob- lems of active and passive immunity to Pneumococcua infec- tion. Progress in the solutiop of this problem has been impeded by two maJor technical difficulties:- one, the diffi- culty of extracting the intact antigen in active form Secause of the ease with which dissociation of the complex occurs; and the other, the difficulty of identifying in such extracts the particular substance or chemo-specific groups which con- fer antigenicity upon the capsular polysaccharide. The firet of these difficulties has been largely overcome, and the second hae been attacked through an indi- rect but promising approach. Following the leads suggested by the work of Perltwelg and of Day, filtered eolutions con- taining the type-epecific antigen in active form have been obtained by extracting the encapsulated cells at an acid re- action with heat, The antigenic activity of the extracts has been tested by injecting them into mice and determining whether the treated animals have acquired specific immunity to infection with pneumococci of the homologous type. In en acid buffer solution (pH B), the antigen reeists heating In a boiling water bath for ten minutes; two injections total- ing 0.4 oc', of a solution containing ak little ae'0,003:mge, N per cc. have proved effective-in immunizing mice againat an homologoua atrain of Type I pneumococcus, The extraction of the antigen, however, is only the first step in the anal- ysis of its chemical nature. Is the type-specific polysac- charldo bound to a protein, or to some other substance? Is it even neces8ary to assume that the effective complex con- siets of a protein coupled to the carbohydrate? This seems 4 f the most reasonable assumption, especially since we havo found that an artifiaial antigen prepared by the coupling of a diazonium derivative of the Type III polysaccharide with animal protein stimulates in rabbits the formation of type- specific antibodies which agglutinate living pneumococci of the homologoua type and protect mice againet infection with virulent Type If1 pneumoqoccl. De6gSte this analogy, may it not be that the.polysaucharide carrying the specific binding group6 is but part of a still larger and more complex carbo- hydrate ~olocule fro: which the antlgenic groups may be eas- ily t&it off, leaving the antigenically inert but specifi- cally reactive polyeaccharide intact. In the case of the pectins for example, partial hydrolysis suffices to split off pectinic acid from the complex and a polysaccharide then remains as a product of hydrolyeie. Aa Well0 point0 out, there ie theoretically no reaeoq why "a carbo.h$dra$e ehould not function as; an an tlgen provided' it is colloidai in ,nature and bf aufflaient noleo*- lar sime! However, to assume that the laok,of antigenicity of the capsular polysaccharidgs is attributable to denature-, tlon in the procese of. isolation ie tb overlook the fact that the.more those eubetancee are chemically purified the more specif lcally reactive they become and the more closely they conform on chemical analysie to the theoretical values for pure carbohydrates. The mere prediction of theoretical possibilities however, is not paramount to the actual identification of the true antigen. The amount of substance required for chem- ical analysis is large and this is difficult to obtain. De- termining whether a given extract contains the effective an- tigen involves the process of immunization and this requires time. It seemed possible, however, that an indirect approach and one which might furnish a clue as to the nature of the substance to be looked for, might be made by determining if possible, what enzyme or class of enzymoe would destrQy the antigenicity of a bacterial extract known to contain the whole antigen. Whether for example, the antigenicity of an extract is destroyed by trypsin, or pegein, etc., or by li- pases or nucleaaes, etc., or whether enzymes, such a6 pecti- nase, emulsion, and others which are known to hydrolize par- tially the more complex carbohydrates would destroy the ef- fective antigen without impairing the specific binding pro- perties of the polyeaccharide. In collaboration with Dr. Goebel this work is now in progress, and the results thus far obtained are promising, but not a6 yet guf~~Ciently.ee~ablished to warrant presenta- tion at this time. ' 1. The Slgnif icance of the &n Teat with Becific `. I P&lyeaccha;ide a6 a Guide to Serum Therapy in Pneymonia. (Dr. prancis~. During the study of the cutaneous reaction of pneu- monia patients to the type specific polysaccharides, it was soon observed that all of the case6 of Type I pneumonia which recovered gave a positive reaction to the Type I polyeaccha- ride; The majority of Type I patients received specific se- rum therapy, while those ouffcring from infections due to Type II and III Pneumococcus do not, Approximately only 50 per cent of the latter group rsacti to the specific soluble substance of the respective types. In none of the patients was this reaction elicit'ed before the onset of recovery, even when type specific therapeutic 6erum had been adminis- tered, Furthermore, in certain instances, when the patient still appeared sick and the problem of additional serum treatment was immediate, a positive skin test was found to indicate recovery and that treatment could be safely discon- t inued. In the case of individuals, in whom no positive re- action Was obtained at any time during the course of the illness, a fatal termination occurred, even though a subsi- dence of fever, sterilization of the blood of the invading organisms and a comparatively high concentration of serum antibodies were observed. Consequently a negative cutaneous reaction to the polyaaccharide was thought to be an indica- tion for continuation of 6erum therapy, The study has beeti extended with the idea of es- tablishing the true value of thle reaction as'a guide to proper serum therapy. At present, data have been obtained in 35 cases of Type I pneumonia. Four of these were not treated with serum but recovered and gave positive skin reac- tions; they serve as controls for the ca6e6 which received specific treatmant. The results are tabulated below. d--e--- ------- --------- --- ----__--__m_____I_ , *Empyema From the table it can be seen that of 26 recovered cases which received serum, only one failed to give a posi- tive response to an injection of the specific polysaccharide and in the exception a purulent complication had developed. In the remaining 25, all of whom gave a positive skin test, recovery took place deepihe suoh complications as sterile pleural effusions, delayed resolution, furunculosia, and re- current acute nephritis. In 5 fatal case6 no skin reaction was obtained at any time. The extended observations have, therefore, con- firmed the impression that the skin test with type specific polyaaccharide is a valuable guide in the 6erum treatment of pneumonia. Ita value lies in the fact that, when positive, it indfcatee that recovery ha6 begun and serum therapy may be safely discontinued, but a negative respoloee is an-indi- . cation that the administration of 6erum should be continued. If, in the face of continued serum therapy with definite clinical improvement a negative reaction persists, a suppur- ative complication should be suspected. If none is found serum therapy should be maintained, although the outlook iS unfavorable. Cb)Q P I* .-k.2 H 119 VI Studies on Natural Resistance and on the fm- -1,,----------------------------------- munlty induced in Rabbits byxuections of R Pnoumococci --- ---_-- -v--p-______v__& 1Dr.----- FrancI& A study is being made of tho natural rosis- tancoe of the rabbit to Type III Pneumococcus and of the form of Immunity produced in rabbits by injsctions of R pneumococci. The method of Robertson and Sia, using serum- leucocyte mixtures, has been shown to be quite sensitive in detecting slight degrees of immunity. For this reason the same mothod, as well as a modification employing whole hopa- rinizod blood, was adopted in the present study. Although tho work is in its early stages, several interesting facts hava been revealed. While the normal rab- bit is resistant to the rabbit-avirulent strain of Typo III Pnoumoco ecus, no pnoumococcidal powers against this organism is demonstrable In the animal's blood. Against the R form of Pneumococcus, however, the normal rabbit oxhibits the power to inhibit the growth of a large number of org8niSmS. The blood of a rabbit containing anti-R antibodies, produced by immunization -with R pneumococci, hae an even greater bat- tericidal power for the R organisms than has the normal ani- ma1 . In addltlon,~.lt possesses a moderate but definite ca-, pacity to prevent the .growth of the avirulent encapsulated Type III Pneumococcus. So far no experiments have been done with the blood of immune rabbits containing type specific antibodies. The state of immunity has been simulated, how- ev`er , by the addition of type specific horse aliti-serum to the blood of the normal rabbit. Under these conditions a `* . marked pneumococcidal effect on the rabbit avirulent Type III pneumococci is also observed, Certain conclusions appear justifiable. The nor- mal rabbit, resistant to the avirulent encapsulated Type III Pneumococcus, shows no evidence in the blood of a phagocytic mechanism for Its disposal. In the R immune rabbit this cn- pacity has been developed to a modorate degree, while the blood of the animal containing type specific immune bodies has a still greater pneumococcidal power. VII. The Principles Governing the &&utinin and -w-m -w-e --- PreclEiGn Reactions. (Dr. Franci& It was observed In the -- -_u_ -I__- course of precipitin reactions that when a prozone was pro- duced with an excess of soluble specific substance of the pneumococcus, the serum had also lost its capacity to agglu- tinate the homologous type-specific organism. This observa- tion ,brought further evidence to Indicate the unity of agglu- tinogen an& preolpltlnogen..,. Ooaeequently the following ex- perimente were .devieed .in an attempt to correlate the meGhan- isms of the .reactione of -agglutination and precipitation. ~0 811 immune aerum -a auapen8ion of the homologous type specific pneumococcus was added. After agglutination had occurred the compact mass was placed in a dilute concen- tration of the specific polysaccharide of the' same type. Under these circumstances preciaitation occurred, with re- moval of the polysaccharide from solutfon, indicating that an excess of antibody was attached to the bacterial cells and was able to unite with additional amounts of antigen, in this case the specific soluble substance. I?0 w , if still more of the free soluble substance was added, the agglutinat- ed organisms became dispersed and remained diffusely suspend- ed in solution. When the bacteria 80 dispersed were removed from the mixture by centrifugation they were found to be in a normal eerological state as shown by the fact that they could be agglutinated again, but only by immune serum for the homologous type of Pneumococcus. Ead ant lbody remained bound to them, the bacteria might conceivably agglutinate spontaneously in heterologoue serum, or in salt solution. This, however, watt not the case. Similarly, organisms which were added to a serum in which a prozone had been created with specific soluble substance were serologically unaltered. The resulta show that the ty?e-specific precipitin and agglut inin react iona are dependent upon the reaction of antibodfea with the same substance; in one case, present In the bacterial cell; in the other, chemically purified anl) separate from the bacteria themselves. The two forma of the type specific substance may be eubatituted for one another without altering the phase of reaction. Heidelberger and Kendall recently studied the pre- cipitin reaction quantitatively and concluded that the var- ious phases of reaction could be expressed in terms of the laws of mas6 action. That is, antibody and specific sub- stance combine in multiple proportions, depending upon the concentration of the two reagents. The present studies con- form qualitatively with theirs, and it seems that the same laws govern both the agglutinin and precipitin reactions. =I. Studies on Epidemiology. (Dr. Stillman). ----- ---- -- 1 Antibody -1--e--- response to immunieation by different routes. ----- ---e-v The work on the epidemiology of pneumonia is being pursued along two lines. Data is being accumulated as to the deve- lopment of immunity following immunization of rabbits with different typee of pneumococci by various routes. The length of time that agglutinins and protective antibodies persist in the serum of rabbits immunized with heat-killed Type I pneumococci has been determined. Agglutinins soon disappeared from the sera of these rabbits. Protective an- tibodiee, on the other hand, could be demonstrated in high concantratione in thoir sera for prolonged periods. It ap- pears that there ie a definite relationship between the to- tal amount of antigen administered and the length of time antibodies may be demonstrated in the blood or rabbits. E'urthermore, there is a close relationship between the route of administration of antigen and the character and persist- ence of the antibody response in Rabbits. 2. Production of experimental P neumonia in ani- -- V-P mals ----.% Work ie aleo in progress on the production of exper- imental lobar pneumonia in laboratory animals. This has a two-fold object. Once the complicated mechanism of the fac- tors necessary to cause an animal to localize pneumococcus ._...~ - ,. infection is understood, we will be able to not only treat the disease of lobar pneumonia in man more efficiently but also know better what precaution8 to observe in order to escape the disease. Although experimental lobar pneumonia has been successfully produced in mice, the various factors entering into the production of pneumonia could not be stud- ied in such a small animal. Eve2 since Wadsworth claimed to have produced pneumonia in partially immunized rabbits in 1904, it has been assumed that pneumonia could be Induced in these animals. It ha8 already been shown that pneumonic consolidation may be produced in partially immunized alca- holized mice by the inhalation method. But we havo been un- able to cause partially immunized rabbits to localize the infection which develops following inhalation of virulent pneumococci. The inability of rabbits to localize the in- fection may possibly be due to the anatomy of the rabbits' lung. Pneumocoool in rabbits rapidly pass through the pleura and cause empyema' ,and pericarditis. There seems to be no at- tempt to confine the infection within the lung Itself. IX. The @tipneumococcus Immune Reactions of -m --a---- Normal Hog~erum. It was reported by Bull and -p (Dr. ICell&,, McKee in 1921 that the blood 8eru.m of normal chickens when injected into susceptible animal8 confers a notable degree of passive antipneumococcus immunity. They also ehowed that the factor responsible for this immunity is associated with serum globulin, and that it is type specific in action. In 1924 Robertson and Sia by means of serun-leuco- cyte mixtures were able to demonstrate the presence of nat- urally occurring antipneumococcus opsonins in the blood of resistant animals. By this technic the serum of normal pig3 showed a high content of these opsonins. Sia in 1929 found that pig 3erum injected intra- peritoneally into mice affords these animals a marked degree of protection against pneumococcus infection. This ant ipneu- mococcua protective action wa8 shown by absorption experi- ments to be type specific in nature. At variance with the widely accepted theory that natural immunity is dependent solely on the cellular defense of the host, are the two examples, here cited, of a natural immunity to pneumococcus infection that is passively truns- ferable through the blood serum, and that appears to be type-specific in nature, The present' study i's an attempt to repeat the ex- periments of Sia, and to further analyze the antipnoumococ- cus immune reactions of the sorum of normal hogs and of other resistant animals. In coafirmation of Sial8 results' it has been found that the serum of normal' hogs when in- jected intraperitoneally confers upon white mice a marked degree of immunity to Pneumococcus, Types I and II. I cc., the optimal protective dO8e of the sorum, usually affords protection against 10,000 to lOO,Om lethal doses of pneu- mococcus culture. The serum yields its maximal protective action when injected four to eight hours before inoculation of the mice with culture. The degree of protection against Type II is consistently higher than that against Type I. The serum gives no protection against pneumococcus infection when injected subcutaneously, Under the same conditions the serum from other animals - rabbit, guinea pig, ox, and sheep, - when injected intraperltoneally, have conferred no protection on mice. The protective principle of hog serum is associ- ated with the serum globulin obtained by precipitation with ammonium sulphat e. Hog serum gradually loses its antipneu- mococcus protective action on standing, so that after two to four months in the ice-box the protective action is entirely, or almost entirely, lost. At room temperature this loss oc- curs more rapidly, In a paraffin-lined tube, under a vase- line seal the serum loees Its protective power in about the same time a8 it does when unsealed. The protective action of the serum against Type I is.loet ueually in shorter time than that againet Type II. The.protectioe power la coneid- erably reduced by heating. the serum to 560 0. for.30 minutes, and is entirely destroyed by ewoeure to a temperature of 64O C. for the earn8 time. The protective action when lost, either through heat, or standing, is not restored to the se- rum by the addition of a small amount of fresh normal serum. The antipneumococcus protective principle for either Type I or Type If can be specifically absorbed. The quantity of pneumococci per volume of serum necessary for specific absorption, is small. The absorption requires only a short time for completion. Absorption of the serum with avirulent R pnoumococcl, and with bactorla othor than pneu- mococci, doos not consistently alter the protective power of the serum. Absorption with charcoal and Kieselgur does not appreciably lower the protective power. Sia in 1922 noted that S pneumococci, when sensi- tized by large amounts of hog serum, show agglutination af- ter separation from the sensitizing serum by centrifugation. It has been found in the present study that hog serum agglu- tinates S pneumococci. The agglutinated pneumococci from a firm, thin disc similar to that produced by type specific ant ipncumococcua serum. In comparison with specific anti- pneumococcus serum large amounts of hog serum are necessary for demonetratlon of the agglutination. It is necessary also, except occasionally In case of Type II, to use Gate's technic for thle demonstration. `The agglutinine for Type II are present in hog serum fn greater concentration, than are those for Type I. Similar to the antipncumococcue protec- tive action In mice, the agglutinlns for S pneumococcl are specifically absorbable and are destroyed by heating the se- rum at 64O C. for 30 minutes. It has also been observed that avirulent R pneu- mococd are agglutinated by hog serum. This aggl utinat ion is of typical R character. The ag.glutinated pneumococci form a granular precipitate leaving the supernatant clear. The maximum R agglutinin titre is ueually 1:250 to 1:500. So far no falling off of the R agglutinin titre has been seen even after the antipneumococcus protective action of the serum Is lost. Although sensitization of living pneumococci with hog serum does not alter their virulence, heat-killed pncu- mococci when sensitized by hog serum provoke a very mcagre specific antibody response in rabbits. With tho concantrations used, no change in colony form has boen brought about by growing either S or R, pneu- mococci in dilutions of hog serum. The R pneumococci cul- tured in presence of the sorum grow in clumps. X. Pathuenesis of Eserimental Intradermal Pneu- -s-- B--w-- ----- ---- mococcus Infection. (Dr. Goodner). The intradermal pneumo- --p--e coccus infection in rabbits offers an experimental disease which bears many analogles to lobar pneumonia. The general nature of this symptom-complex has been studied-for a num- ber of years and is now suff id ently well understood to serve as a basic method for more specific proJacts. Faith Dr. Rhoade the histological pathogenesis of this lesion has been studied and found to be comparable to that of lobar pnoumonia in man. The findings are described in detail in another section. The outstanding feature of the early lesion is the edema fluid: its acciamulation occurs before any change in the cellular picture, and in the pro- ---._. %-- - gressing or moving lesion its ;>assnge tLrough the tissue precedes any other sign of reaction between tissue and micro- organ ism. It seems probable that the advancing fluid car- ries with it the first. infecting organisms and consequently inoculates all tissues which it reaches. The resultant in- fection appears to be duo. not to an active invasion, but rather to a progressive passive inoculation. Edema fluid has been removed from the developing lesion and its propertios studied in vitro. obtained If --- free from blood and tissue elements such fluid does not clot even after long standing. That this property is not due to a deficiency of fibrinogen is shown by the fact that a firm clot promptly appears on the addition of thromhoplastin. Evidence points to a high antithrombic content since the edema fluid has the property of retarding the clotting of normal rabbit blood. The clotting time of the blood in these rabbits infected with pneumococcl is prolonged, par- . . . A.'. t titularly in severe casee, but the effect Is never as pro- , <. j _ . . `. -*, " I ._ .i -I nounced as in the exudative fluid where the concentration of the antithrombic factor,appears to be much'greater. The na- . ture and 8ource of this? antithrombid substance are now being ,.I . . . . . . . _j I i- investigated. . ,I A study has been made of the factors which.have to do with the development, movement, final localization, and extent rf the dermal lesion in the rabbit. The direction of movement is determined by gravity and is independent of the ---__ _-.----.. distribution and course of blood vessels and lymphatics. The lesion seems always to seek to reach the more directly dependent area. The direction of movement may be modified by changing the position of the animal or by arrrmging ar- tificial guiding structures such ae scars. If the skin of the abdomen is the final site of localizatj.on there is a massive collection of edema fluid. Such edematous lesions are not obtained in areaa where the skin is tense, such as over an extremity. Drainage distance largely determines the volume of edema fluid accumulating In a given area. Numerous factors modify the development and rate of movement. Perhaps the most striking of those yet obeerv- ed is the accelerating or synergistic co-infection with & &nf luenzae. This seems of particular significance since v- some form of the Pfelffer bacillus is found in a large per- centago of sputa from cases of lobar pneumonia. If B. in- fluenzae!and pneumococci (Type I) are inJected together in- tradermally ,the effect- iS a marked acceleration in the de- velopment and movement of the lesion. The lag period of the pneumococcic infection 16 almoet entirely eliminated, Other studies on the associative relationships of these organisms are now in progress. I Intradermal Infection with a Rabbit Virulent Type -+--me ------------- III Pneumococcua.-@r. Goo'dnerL The character of tke in- --- ------ -------- tradormal infection nith:a rabbit virulent strain of Type III pneumococcus has been studied and found to follow essen- _..--___ . ,... * tially the same course as with Type I, although the disoasc is much more severe and tho fatality rate higher. c3sos terminated more often by lysis than by crisis. This is pos- sibly explained by the low grade activa immunity which fol- lows artificial immunization or recovery from tho disease. Bocoverod animals show but slight resistance to reinfection even at short intervals. The immunity acquired from a course of the disease ia of sufficient quantitative chnractor to prevent death after reinfection but not sufficient to pru- vent a severe infection. The use of Type III antiserum (by oursolf and by others)in treating experimental infections has yieldod poor results. A selected serum was concentrated by our method and al though high agglutinin and precipitin titers were ob- tained, this concentrate fails completely to protect mice. It haa, however, a favorable influence in the rabbit infec- tion, although even in large amounts it seema incapable of promptly terminating the disease. The enzyme which specifically decomposes the cap- sular polyaaccharide of Type III pneumococcus is being uzed. in treating this experimental disease. Preliminary experi- ments have given encouraging results but many factors are as yet not suffidently understood to warrant final conclusions. The enzyme preparations as obtained at this time possess a primary toxicity for normal rabbits and are particularly toxic for infected animals. With certain preparations which showed the lower range of toxicity the therapeutic effect was marked. The blood stream became free of organisms and after an initial rise the temperature dropped to normal lcv- els. Repeated injections of the enzyme are necessary to prevent relapses. XI. Study of the Transformation of the Specific -----------------Me- Types of Pneumococcus. (Dl-. Alloway). The experiments car- ried out by Griffith in 1928 for the first time brought con- vincing evidence that specific types of Pneumococcus are not absolutely fixed but may, under special conditions of growth, be transformed one into another. It had been known previous- ly that type-specific pneumococcl may be degraded into the so-called common R forms, but it was generally believed that, if, these forms ever again reverted to type, they alrvays re- verted to that type from which they were originally derived. Griffith brought about the transformation of types by inoculating mice subcutaneously with small quantities of living R organisms together with large quantltfes of heat- killed S cells of a type other than that from the R strain ,' originally came. Under these conditions, the R forms resum- ea the specific .prdpertiea. of `the particular $ hype uaed'in" . . the heated inoculum. In other words, the process of r&&r: sion may now be specifically oriented at will, depending upon the type of Pneumococcus used in the reaction-mixtures. Dawaon in 1930, working in this laboratory, con- firmed Griffith's results and later defined the cultural conditions under Which transformations similar to those orig- inally induced in the animal body may now be brought about in the culture tube, Pncumococci which have assumed the specific charactors of the new type in viva ------ 9 show no tcnden- cy to rovort spontaneously to the original type. With the acquisition of a new specificity, the R forms also regain the property of virulonco, a quality usually not restored by mere cultivation on artificial media. At present further studies are In progress with the hope of finding out the basic factors responsible for these remarkable changes in biological specificity. Attempts are being made to extract from type specific pneumococci, the substance, or substances, responsible for the actlvition of the Ii forms. Since the type-specificity of pneumococci is dependent upon the pre- sence of the capsular golysaccharide, this substance in pur- ified form was used instead of the whole celle. The failure to induce the change under these conditions indicates that the speclf So polysaccharide, in a chemically purified etate, does not, !.,by itself, fanction'ae the activating substance. Reference.h~a.:already b88n made. in ,thls report; to a method of extracting the oapaular polysaccharide yin `a form inwhich it still retains its antigenic property. Since acid oxtrac- tion with heat has boon found to remove from the cells this i otherwise easily dissociated antigen, the same method waa used in attempts to extract in active form the bacterial substance which is concerned in the process of transformation. Extracts prepared in this way, from which formed cells were removed by prolonged centrifugation, have proved active in causing R pneumococci derived from Type II to as- sume the specific characters of the Type III S cells from which the extract was made. However, when the same extract was passed through a Berkefield filter it lost its activity and was no longer effective in bringing about the chunge. Although too little is known of the biologically active sub- stances which appear to be specific activators of the R forms of pneumococci, the possibility exists that they may function as a co-zymase or activator of a zymase which exists in an inactive state in all R cells. This hypothesis fur- nishes at least a working basis and seems the more likely since it is now apparent that every R cell has potentially the capacity to synthesize any one of the type-specific cap- sular polysaccharidee, which one it happens to elaborate be- ing dependent ,upon a apeoific stimulus, ,or activator. Varloue methods of ,ertraction are being.tried with the hope of obtaining the &active substance from the. type: .: specific cells. _ From suuh extracts it ,may then be possible I _ to isolate the active principle and thus to obtaPn a more definite knowledge of its chemical and biological properties and its relation to the mechanism of this important phenomenon. XII Simnificance of Oxidation-Reduction Processes -AA I-w_L- - ---- ---_I -----m---m in Bacterial Growth. (Dr. Dubos). When Pneumococcus is seed- --- --- ---- ed into plain broth, it is necessary to use a fairly large inoculation in order to insure the initiation of growth, The work of previous years had established that this "bat- teriostatic" action of the broth, as expressed by the lack of grotyth of very small inocula, can be corrected by a var- iety of treatments Thich have in common the development of reducing conditions in the medium. It has now been shown that the bacteriastatic substance is present in the peptone used in the preparation of the broth in which this substance usually exists in an oxidized form. It appears furthermore that this bacteriastatic substance can be reversibly oxidix- ed and reduced and that the bacteriostatic properties aro oxhibited only by the oxidized form, and not at all by the reduced form. In this respect, it behaves like methyleno blue, gallocyanlne and the various indophenols, dyos which have been shown previously to be bacteriostntid only in the oxidised form. Different brands of commercial peptonos contain varying amounts of bactorioetatlc substance.. lhie substance, although its nature, is not known, can be titrated by.,means of reducing substances, in particular the reduced thio-acids. The principle of the titration ie to determine what amount of the reduced substance la necessary to correct the toxic- ity of a certain amount of the bacteriostatic material. Such titrations have revealed that the bacteriostatic action can be recognized with concentrations corresponding to as little as 10 -5 molar concentration of thioglycollic acid. These concentrations are of the order met with in catalytic reactions and one may wonder whether the bacteriostatic sub- stance of the peptone does not act as anticatalyst in some phase of the metabolism of the cell. It was of obvious interest to attempt to obtain pcptone preparations free of this bacteriostatic action. This has boon achiovod by two completely different methods, A, Purification of a brand of commercial poptone. -B-c- ---- .- I__ It has been found possible to remove 90-95 per cent of the bacteriostatic fraction of Fairchild peptone by procipitn- tion with concentrated hydrochloric acid. The bacteriosta- tic substance thus precipitated appears as a pigment which can be reversibly oxidized and reduced. In the course of this process, the color of the material changes from green to brown; the bacteriostatic action is associated with the green (oxidieed) form. AEI mentioned pr'eviously, the acid precipitation removes only 90-95 per cent of the bacterioetatlc material,' Another bacterioatatlc fraction is associated with the ace- tone soluble component of the peptone. A purified peptone was prepared by removal of these two fractions. With this purif led preparati on, a medium could be prepared in which a positive growth of Pneumococcus was obtained, in the absence of blood or any reducing substance, with an inoculum as small as .000,000,1 cc, of culture. - -------- a i3. Preparation of peptones from Eure uroteins. ---- ----__--- - ---------- ----i-------- There are indications tilat the bacteriostatic substance is darivcd from some constituents of the animal tissues usad in the preparation of tho poptonas, and it was thought that peptones prepared from purified proteins nould bc devoid of bacterioatntic substance. In fact, pcptonos prepared by peptic or tryptic digestion of caeoin have justified this assumption. These caseln digests, when used for the preparation of llplnin broth" give media in which the groi7th of Pnoumococcus can bo initiated with very amall inocula; it has also beon ob- sorvod that the quantity of growth, as moasured by t;le num- ber of cells or amount of bacterial protein synthetizcd, is much highor with these caeein digests than with any of the ordinary peptonaa which have been tosted. IYork is in pro- gress establishing the value of these peptones for the growth of other Gram positive cocci, of B.`granulosis, of the anae- robic filter passing group, and of strict anaerobea. It may be mentioned that Dr. A.' R. Doch es has used these preparal: tione in experiments on the cultivation of virue of the com- mon cold An vitro. -m-v Equally satisfactory results, although not so com- plete, have been obtained with peptone prepared from other purified proteins (fibrin, globin). XIII. B i b 1 i o g-r aJ,&-& ---e-----v--- -- Avery, O.T., and Dubos, Rene: The specific action of a bac- terial enzyme on pneumococci of Type III. Science ----- 1 1930, 22, 151. Avery, O.T., and Dubos, Rene: The protective action of a specific enzyme on Pneumococcus Type III infection in mice. LIn manuscriPt1. -v---L- - - Babers, F.H., and Goebel, W.F.: The molecular size of the Type III specific polysaccharide of Pneumococcus. J. ai Chem ----,,I--d-' lxxxix, 1930, 387-394. Dubos, Rene: The bacterioetatic action of certain components of commercial peptones as affected by conditions of oxidation and reduction. J. Exaer Ved -----h-LI--* 8 1930, 52 --* 331-345. Dubos, Rene; and Avery, O.T. lar polysaccharide bacterial enzyme. : The decomposition of the capeu- of Type III Pneumococcus by a fi manuscripti. ------ Julianelle, L.A.: The distribution of Friedlinder~s bacilli of different types, J. Expar. Ided., 1930, 52, 539.. -a Rhoads, C.P., and Goodner, K.: On the pathogeneais of exper- imental dermal pneumococcus Infection in the rab- bit. un m~uscriptl. -- Stillman, E.G.: The effect of the route of immunization on the immunity response to Pneumococcus Type I. J. Exper. Ked., 1930, 51, 721-727. Stillman, E.G.: Susceptibility of rabbits to infection by the inhalation of Type II pneumococci, J. Expsr. )&I., 1930, 52, 215-224. Francis, T.,Jr., and Tillett, 8.6.: Cutaneous reactions in Pneumonia. The development of antibodies follow- ing the intradermal injection of type specific polysaccharide, J. Exper. Med., 1930, 52, 573-585. --- Goebel, W.F.: The preparation of the type-specific polysac- charidee of Pneumococcus. J. Biol Chem ----&-A) 1930, lxxxix, 395-398. Goodner, K., and Rhoads, C.P.: Factors involved in the de- velopment and localization of the dermal pnoumo- coccic lesion in the rabbit. & manuscript),. w-u-- Stillman, P.G.: Development of agglutinins and protective antibodies in rabbits, after inhalation of T~rrpe II pneumococci, J. Sxper.-ge$., 1930, 52, 225. --- Stillman, E.G.: Duration of demonstrable antibodies in serum of rabbit6 immunized with pneumococci Type I, IIn manuscripti. Tillatt, W.S., and Francis, T.,Jr.: Serological reactions in pneumonia with a non-protein somatic fraction of Pneumococcus. J. Exper. Med. ---s-m * 1930, 55, 561-571, and J Clin Invest A-&--de--- * 1930, 2, 11. Tillett, W.S., Goebel, W.F., and Avery, O.T.: Chemical and irr,munological properties of a species-specific carbohydrate of pneumococcl. J. 2xper. Med,, 1930, a, 895-900.